ArticleMolecular brain2021
Optimization of protocols for pre-embedding immunogold electron microscopy of neurons in cell cultures and brains.
Article in Molecular brain, 2021. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 21 papers.
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Who cites it
21 citing papers in PubMed.
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- ERG phase separation attenuates cellular senescence.iScience · 2026Article
- Transmission electron microscopy of cultured cells: Optimized protocols for ultrastructural studies and post-embedding immunogold labeling.Biology methods & protocols · 2026Review
- Advances in multiscale myelin imaging: from classical histology to functional insights.Frontiers in cellular neuroscience · 2026Review
- Review
- Immunoelectron microscopy: a comprehensive guide from sample preparation to high-resolution imaging.Discover nano · 2025Review
- Age-dependent integration of cortical progenitors transplanted at CSF-neurogenic niche interface.Frontiers in cell and developmental biology · 2025Article
- Ultrastructural characterization of hippocampal inhibitory synapses under resting and stimulated conditions.Molecular brain · 2024Article
- The NFATc1/P2X7 receptor relationship in human intervertebral disc cells.Frontiers in cell and developmental biology · 2024Article
- High iron in mouse olfactory ensheathing cells at the glia limitans in the olfactory bulb underlies MRI TImaging neuroscience (Cambridge, Mass.) · 2024Article
- An overview of the detection of bovine respiratory disease complex pathogens using immunohistochemistry: emerging trends and opportunities.Journal of veterinary diagnostic investigation : official publication of the American Association of Veterinary Laboratory Diagnosticians, Inc · 2024Review
- Electron Microscopy of Neurons on Biomimetic Substrates.Methods in molecular biology (Clifton, N.J.) · 2024Article
- Rehydration of Freeze Substituted Brain Tissue for Pre-embedding Immunoelectron Microscopy.Microscopy and microanalysis : the official journal of Microscopy Society of America, Microbeam Analysis Society, Microscopical Society of Canada · 2023Article
- ER-PM Junctions on GABAergic Interneurons Are Organized by Neuregulin 2/VAP Interactions and Regulated by NMDA Receptors.International journal of molecular sciences · 2023Article
- Unorthodox localization of P2X7 receptor in subcellular compartments of skeletal system cells.Frontiers in cell and developmental biology · 2023Article
- Bringing synapses into focus: Recent advances in synaptic imaging and mass-spectrometry for studying synaptopathy.Frontiers in synaptic neuroscience · 2023Review
- Modification of the synaptic cleft under excitatory conditions.Frontiers in synaptic neuroscience · 2023Article
- Function of ceramide transfer protein for biogenesis and sphingolipid composition of extracellular vesicles.Journal of extracellular vesicles · 2022Article
- Current methods to analyze lysosome morphology, positioning, motility and function.Traffic (Copenhagen, Denmark) · 2022Review
- Palmitoylation of A-kinase anchoring protein 79/150 modulates its nanoscale organization, trafficking, and mobility in postsynaptic spines.Frontiers in synaptic neuroscience · 2022Article
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4 authors.
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Abstract
Immunogold labeling allows localization of proteins at the electron microscopy (EM) level of resolution, and quantification of signals. The present paper summarizes methodological issues and experiences gained from studies on the distribution of synaptic and other neuron-specific proteins in cell cultures and brain tissues via a pre-embedding method. An optimal protocol includes careful determination of a fixation condition for any particular antibody, a well-planned tissue processing procedure, and a strict evaluation of the credibility of the labeling. Here, tips and caveats on different steps of the sample preparation protocol are illustrated with examples. A good starting condition for EM-compatible fixation and permeabilization is 4% paraformaldehyde in PBS for 30 min at room temperature, followed by 30 min incubation with 0.1% saponin. An optimal condition can then be readjusted for each particular antibody. Each lot of the secondary antibody (conjugated with a 1.4 nm small gold particle) needs to be evaluated against known standards for labeling efficiency. Silver enhancement is required to make the small gold visible, and quality of the silver-enhanced signals can be affected by subsequent steps of osmium tetroxide treatment, uranyl acetate en bloc staining, and by detergent or ethanol used to clean the diamond knife for cutting thin sections. Most importantly, verification of signals requires understanding of the protein of interest in order to validate for correct localization of antibodies at expected epitopes on particular organelles, and quantification of signals needs to take into consideration the penetration gradient of reagents and clumping of secondary antibodies.
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